Generation of surface coating diversity

ABSTRACT

The present invention relates to a surface discovery system comprising chemical compositions and high-throughput combinatorial synthesis methods for generating large numbers of diverse surface coatings on solid substrates. This surface discovery platform is built upon a fundamental chemical unit refereed to as a synthon. Each synthon comprises at least three elements: a chemical backbone coating on the solid substrate that comprises a passive (P) constituent and an active (A) constituent; a spacer unit (S) separating the backbone from a functional group; and a functional group (F). Variation of these synthon elements allows generation of large libraries surface coatings with a broad range of molecular and macroscopic properties. Further the spectrum of surfaces provided by the invention permits optimization of the wide range of solid-phase applications that involve surface immobilization of molecules.

FIELD OF THE INVENTION

The present invention relates to surface coating technology. Inparticular, the invention relates to a method for generating a libraryof different surface coatings on a substrate, to a method for optimisinga substrate surface for a solid phase application and arrays or beadspossessing discrete regions of particular optimised surface coatings.

BACKGROUND OF THE INVENTION

Current surface coating technology provides a relatively limited numberof established surfaces that may be used in new solid-phase chemical orbiochemical applications. The lack of established surfaces stemsprimarily from the difficulty associated with the generation ofdifferent surface coatings. While large numbers of chemically diversecompounds may now be generated in solution without too much difficulty,the ability to graft these molecules on to a solid phase and create alarge number of surface coatings has proven a much more difficultproblem to solve. In particular, the chemistry of grafting moleculesonto solid phases to create surface coatings is highly unpredictable,and has to date remained more an art than a science.

There are numerous applications where a diverse range of novel surfacecoatings would be particularly advantageous, for example in the area ofsolid phase biological assays. With the number of novel proteins growingeach day, there is growing need for novel solid phase surfaces that arecompatible with the immobilization of these complex macromolecules.Despite this need, in practice there are to date relatively few solidsurfaces available across the wide range of solid phase applicationsused to study biological molecules. For example, in the area of captureand display of biomolecules each commercial supplier has its ownparticular solid phase surface embodiment that is prescribed across abroad range of specific applications. One specific example is a surfacegenerated using the well-established PEG chemistry as described in anarticle by Ruiz-Taylor et al. (“Monolayers of derivatizedpoly(L-lysine)-grafted poly(ethylene glycol) on metal oxides as a classof biomolecular interfaces,” PNAS USA 98: 852-857 (2001)). Anotherexample is the relatively new boronic acid complex chemistry used toprepare surfaces for immobilization of proteins described by Stolowitzet al. (“Phenylboronic Acid-Salicylhydroxamic Acid Bioconjugates. 1. ANovel Boronic Acid Complex for Protein Immobilization,” BioconjugateChemistry 12: 229-239 (2001)).

Surface plasmon resonance (SPR) has now been widely adopted as atechnique for detecting protein-ligand and protein-protein bindinginteractions. However the utility of SPR with a particular proteinsystem depends greatly on the vagaries of how that macromolecule bindsto the surface of the solid substrate when immobilized. If a particularSPR surface causes a protein of interest to bind in an orientation thatis unfavorable for detecting ligand binding, there are only a handful ofalternative surfaces with a limited range of binding properties fromwhich to choose (see, e.g. Rich and Myszka “Advances in surface plasmonresonance biosensor analysis,” Current Opinion in Biotechnology 11:54-61 (2000)).

Similarly, mass spectrometry also is now widely employed for theanalysis of biological macromolecules. These methods typically involveimmobilization of a protein on a surface of substrate where it is thenexposed to a ligand binding interaction. Following ligand binding (ornon-binding) the molecule is desorbed from the surface and into aspectrometer using a laser (see, e.g. Merchant and Weinberger, “Recentadvancements in surface-enhanced laser desorption/ionization-time offlight-mass spectrometry,” Electrophoresis 21: 1164-1177 (2000)). As inthe SPR experiment, the success of the mass spectrometry experimentdepends largely on the interaction between the immobilized protein andthe surface. In view of the thousands of proteins with different surfaceinteractions, there is clearly a need for a large number of differentsubstrate surfaces in order for mass spectrometry to be appliedsuccessfully to the high throughput analysis of the proteome.

Accordingly, the inability to provide a diverse array of surfacecoatings stands as an impediment to development in solid phasebiological technologies such as biological assays and diagnostics, andbiomaterials. Such an impediment also extends across a broad spectrum ofother technologies, ranging from solid-phase chemical synthesis,catalysis development and separation and purification technologies.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of generating alibrary of different surface coatings on a substrate comprising:

-   -   a) selecting a surface coating synthon of formula B—S—F, wherein        B is a copolymer of at least one passive constituent P and at        least one active constituent A, S is a spacer unit and F is a        chemical or biological functional group, wherein S is attached        to an active constituent A of copolymer B, and wherein the        synthon has at least one point of diversity selected from P, A,        S and F;    -   b) applying backbone coating(s) of the selected copolymer B onto        a substrate;    -   c) attaching the selected combination(s) of spacer unit S and        functional group F to an active constituent A of copolymer B        according to said selected synthon;    -   wherein steps b) and c) are performed such that surface coatings        according to the synthon are generated on localised regions of        the substrate, thereby providing said library of different        surface coatings on the substrate.

In another aspect,.the present invention provides a method of optimizinga substrate surface for a solid-phase application involvingimmobilization of a molecule comprising:

-   -   a) generating a library of different surface coatings on a        substrate by a method comprising:        -   1) selecting a surface coating synthon of formula B-S-F,            wherein B is a copolymer of at least one passive constituent            P and at least one active constituent A, S is a spacer unit            and F is a chemical or biological functional group, wherein            S is attached to an active constituent A of copolymer B, and            wherein the synthon has at least one point of diversity            selected from P, A, S and F;        -   2) applying backbone coating(s) of the selected copolymer B            onto a substrate;        -   3) attaching the selected combination(s) of spacer unit S            and functional group F to an active constituent A of            copolymer B according to said selected synthon;        -   wherein steps 2) and 3) are performed such that surface            coatings according to the synthon are generated on localised            regions of the substrate, thereby providing said library of            different surface coatings on the substrate;    -   b) exposing at least two of the surface coatings in the library        to the molecule to be immobilized; and    -   c) determining which of the at least two surfaces results in        better performance of the immobilized molecule in the        solid-phase application.

In a further aspect, the present invention provides a biologicalmolecule detection unit capable of detecting at least two biologicalmolecules, said unit comprising a substrate having a plurality ofsurface coatings wherein at least two of said coatings are different,and tailored to recognise, bind to or associate with a particularbiological molecule. A person skilled in the art would be able to adaptthe methods described herein to prepare such a detection unit.

The present invention provides a method for generating a library ofdifferent surface coatings on a substrate which can be advantageouslyused as part of a surface discovery system. The library is generatedusing a unique synthon approach that provides an architectural frameworkfrom which the specific surface coatings can be realised.

The present invention fills a critical gap in solid surface technologyby providing a high-throughput platform for the rational generation andexploration of surface coatings with novel molecular and macroscopicproperties. The diverse combinatorial libraries of surface coatings thatmay be generated in a high-throughput manner using the synthon-basedapproach disclosed herein may be applied across a broad spectrum oftechnologies, ranging from solid-phase chemical synthesis, catalysisdevelopment, separation and purification technologies, biological assaysand diagnostics, and biomaterials development.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The Synthon

As used herein the term “synthon” is used to refer to a fundamentalchemical unit, or building block, which provides an architecturalframework to design and develop a diverse array of surface coatings on asubstrate. The synthon comprises three basic elements and cansimplistically be represented as B—S—F, wherein B is a copolymer of atleast one passive constituent P and at least one active constituent A, Sis a spacer unit and F is a chemical or biological functional group. Thespacer unit S is attached to an active constituent A of copolymer B, andthe synthon has at least one point of diversity selected from P, A, Sand F.

Together, the space unit and the functional group form a “functionaltether” that may be modified further with chemical entities. Simplecombinatorial chemical variation of the four points of diversity (i.e.passive constituent, active constituent, spacer unit, and functionalgroup) of the synthon described above allows one to generate potentiallythousands of unique but related surfaces. Systematic variation of theactive constituent, passive constituent, spacer unit and functionalgroup allows generation of libraries of different surface coatings thatspan a spectrum of microscopic and macroscopic properties. Theselibraries of surfaces may be further explored using a variety ofanalysis techniques to discover the optimal surface for a variety ofapplications. Consequently, the synthon-based approach to generatingsurface coating diversity described herein provides a platform akin tocombinatorial synthesis of small molecules and peptide libraries.

Although combinatorial approaches to generating molecular diversity havebeen employed to generate new lead compounds in the drug discoveryprocess, these strategies have not to date been employed in the searchfor novel surface coatings that exhibit advantageous properties. Indeed,the standard solid phase combinatorial chemistry approaches used in drugdiscovery focus on generating variety in the small molecule propertiesand avoid diversity in the solid phase to which it is attached. Thesolid phase is viewed simply as a convenient handle to be disposed ofafter cleavage of the small molecule. Consequently, there has beenlittle systematic exploration of solid phase surfaces and how theirproperties may be varied to optimize solid phase applications.

Together, the space unit and the functional group form a “functionaltether” that may be modified further with chemical entities. Scheme 1below illustrates a more detailed representation of a potentialstructure of the synthon.

In scheme 1, the synthon further comprises a control agent C which maybe optionally attached to copolymer B, as represented by —[P-A]-. Thecontrol agent C may be used as a means to prepare copolymer B underliving/controlled polymerization conditions, or alternatively as a meansto modify copolymer B. Preferred control agents include, but are notlimited to, RAFT control agents, ATRP control agents, and nitroxidecontrol agents. The use of a control agent advantageously provides ameans to carefully control and design the molecular architecture ofcopolymer B, for example by controlling molecular weight distributionand/or distribution of monomeric units within the copolymer chain.

Simple combinatorial variation of the four points of diversity (i.e.passive constituent, active constituent, spacer unit, and functionalgroup) that form the basic synthon described above allows one togenerate potentially thousands of unique but related surfaces. In onepreferred embodiment, the diversity is derived solely from the spacerunit S. In another preferred embodiment, the diversity is derived solelyfrom the functional group F. In yet another preferred embodiment, thediversity is derived from both the spacer unit S and the functionalgroup F.

In a relatively simple example, starting with one backbone coating on abase material (i.e. where the P and A constituents are kept constant)treatment with at least ten spacer unit S variants, and 10 differenttransformations of the functional group F, results in 100 differentsurfaces.

Of course, greater numbers of diverse compounds may be achieved if acontrol agent C is incorporated as another point of diversity. Thecontrol agent may be used as the start site for living-controlledpolymerization reactions. Consequently, the backbone coating may bemodified by living-controlled polymerization independent ofmodifications at the spacer attached to the active constituent of thebackbone.

Additionally, diversity may be achieved by utilizing orthogonal reactionstrategies and/or combining mixtures of elements in building thesynthons. For example, the passive constituent may act as a secondactive constituent by modifying it using a reaction orthogonal to thatused to modify the first active constituent. Consequently, in someembodiments both the active and passive constituents may be modifiedwith spacers to generate greater surface diversity.

Advantageously, the present invention allows construction of librariescomprising preferably at least 10, more preferably at least 100, stillmore preferably at least 1000, most preferably at least 10,000 differentsurface coatings.

Preferably, the library in accordance with the present invention isprepared in a multiplex format, and the library is also used in amultiplex format.

The Backbone Coating and its Parameters

The present invention involves applying backbone coating(s) of theselected copolymer B onto a substrate. The backbone coating provides themacroscopic design element in the method and is preferably covalentlybound to the underlying substrate. In a preferred embodiment, thebackbone coating is bound to the underlying substrate through well-knownmethods of polymer grafting, or other methods of coating a solidsubstrate such as dip coating, plasma polymerization, vapor deposition,stamp printing, gamma irradiation, electron beam exposure, thermal andphotochemical radiation.

As the backbone coating, copolymer B comprises at least one passiveconstituent P and at least one active constituent A. These constituentsmay be viewed as monomeric units within the copolymer B. The copolymer Bmay also comprise other monomeric units. In some embodiments, thebackbone coating may comprise more than one active and more than onepassive constituent. As described in greater detail below, the activeand passive constituents may be selected from a wide spectrum ofcompounds well-known in the art. Preferred are those compounds amenableto grafting or other methods of coating a solid substrate (e.g. dipcoating, plasma polymerization, vapor deposition, stamp printing, gammairradiation, electron beam exposure, thermal and photochemicalradiation).

Generally, the backbone coating may be attached to the underlyingsubstrate through either the active or passive constituent. In someembodiments, both constituents may engage in bonding interactions withthe substrate.

The Active Constituent

The role of the active constituent is to provide a point for futurediversity and would be represented by a functional group that is wellknown in the art to under go a vast number of chemical transformations,such as an amine, hydroxyl, anhydride, ester, carboxylic acid, ketone,epoxide, isocyanate and so on. Many well-known chemical monomers may beemployed as active constituents in the formation of a synthon backbonecoating. Selection of a particular set of active constituents may dependon the passive constituents selected and the desired chemistry forapplying the backbone coating to the substrate.

Generally, the active constituent comprises a chemical moiety, orsubstituent group that may be chemically modified with a spacer compound(see described below).

For example, in an embodiment where gamma-initiated free-radicalgrafting is employed, one could employ any of the following monomers asthe active constituent in the backbone coating: hydroxyethylmethacrylate, maleic anhydride, N-hydroxysuccinimide methacrylate ester,methacrylic acid, diacetone acrylamide, glycidyl methacrylate, PEGmethacrylate.

In an alternative embodiment, more than one different active constituentmay be present in the same backbone coating. For example, the coatingmay be made using a mixture of two active monomers. Once prepared, usingwell-known orthogonal approaches to chemical transformations, it ispossible to differentially modify each of the different activeconstituents in the presence of the others, in a sequential andpredetermined manner.

In preferred embodiments the active constituent comprises a chemicalmoiety, or substituent group that is amenable to surface graftingmethods known in the art.

Table 1 below lists an exemplary selection of chemical monomers that maybe used to provide the active constituents in the present invention. Thecompounds in this table are not intended to be limiting. Many commonchemical variants of these compounds, as well as, other compounds notlisted here but well-known in the art of surface modification may alsobe used.

Preferably, copolymer B comprises an active constituent A derived fromthe polymerised residue of maleic anhydride. TABLE 1 Selection of ActiveConstituents ACTIVE 1 2 3 4 A

B

C

D

The Passive Constituent

Whereas the active constituent acts primarily as the point of attachmentof the spacer, the primary role of the passive constituent ismodification of molecular or macroscopic environment of the surfacecoating. For example, a set of passive constituents may be selected thatmodify the charge or the hydrophilicity of the surface coating.Modifications to passive constituents in a three dimensional stablenetwork forming a surface coating allows determination of optimalsurface properties for solid-phase applications. For exampledetermination of a surface that allows binding of non-contiguousepitopes of a biomolecule so that they are available for a bindingassay.

Further, the passive constituent also may act as a spacer unit for theactive composition of the coating, in order to distribute the activegroup alternating, randomly, statistically or in a gradient fashionthroughout the coating.

As mentioned above, in some embodiments the passive constituent mayserve double-duty, also acting as a second active constituent forattachment of a spacer unit. Consequently, in some embodiments thepassive constituent comprises a chemical moiety, or substituent groupthat may be chemically modified with a spacer compound. In thisembodiment the reaction conditions for modification of the passiveconstituent are preferably orthogonal to those used to attach a spacerto the first active constituent.

The chemistry of the passive constituent may be provided by well knownchemical monomers (preferably those that are commercially available)such as: styrene, dimethyl acrylamide, acrylonitrile, N,N dimethyl (ordiethyl) ethyl methacrylate,2-methacryloyloxy-ethyl-dimethyl-3-sulfopropyl-ammounium hydroxide, andmethoxy PEG methacrylate. Preferably, copolymer B comprises a passiveconstituent B derived from the polymerised residue of styrene.

In preferred embodiments the passive constituent comprises a chemicalmoiety, or substituent group that is amenable to surface graftingmethods known in the art.

Table 2 below lists a selection of chemical monomers that may be used toprovide the passive constituents of the present invention. The compoundsin this table are not intended to be limiting. Many common chemicalvariants of these compounds, as well as, other compounds not listed herebut well-known in the art of surface modification may also be used.TABLE 2 Selection of Passive Constituents Passive 1 2 3 4 5 A

B

C

D

In an alternative embodiment, the desired macroscopic property of asurface coating for a selected solid phase application may be derived byin silico analysis of a range of synthon structures. Based on the insilico results, a passive constituent monomer with the chemical featuresnecessary to generate the macroscopic property may be synthesized.Alternatively, the appropriate chemical features of the passiveconstituent may also be derived by in situ chemical transformation of analready applied backbone coating. In preferred embodiments, such in situtransformations of the passive backbone constituent are carried out inan orthogonal reaction scheme in order to maintain the integrity of theactive constituent.

Application of the Backbone Coating

Generally, the synthon backbone coating may be applied to the substrateusing any of the vast assortment of surface modifications methodspresent in the art (e.g. dip coating, plasma polymerization, vapordeposition, stamp printing, gamma irradiation, electron beam exposure,thermal and photochemical radiation).

In one embodiment, the backbone coating is polymerized from theconstituent monomers on the solid substrate using chemistry well-knownin the art. A wide range of polymerization processes present in the artmay be utilized. For example, controlled and/or living polymerizationtechniques of cationic, anionic, radical (such as NMP, ATP, ATRP, RAFT,Iniferter), condensation, and metathesis (such as ROMP and ADMET) allmay be used. Non-controlled methods of polymerization well known in theart may also be utilized with this invention.

In one preferred embodiment, the backbone coating may be provided bymethods known to afford living polymerization. By definition, the endgroups of such living polymers have the ability to be furthertransformed, either by addition of a monomer to extend the macromoleculewith the same monomer, a mixture of monomers or new monomericcompositions. Also, the end groups may be modified using any of avariety of organic chemistry transformations well-known in the art ofsmall molecule manipulation.

In embodiments where the synthon includes a control agent (C) end groupon the backbone, living-controlled polymerization may be used to furthermodify the backbone coating. Control agents and methods of conductingliving-controlled polymerization are well-known in the art. Methods ofliving-controlled polymerization and re-initiation on the surfaces ofnon-functionalized solid substrates is described in co-pending U.S.patent application Ser. No. 10/109,777 filed Mar. 28, 2002. Also, see,e.g. Canadian Patent applications 2,341,387 and 2,249,955 which disclosemethods of living-controlled polymerization on solid polymer substrates.

Alternatively, the backbone coating may be applied to the substrate as apolymer solution, comprising macromers that will allow tethering bycomplementary chemistry to the surface of the substrate or encourageentanglement of the polymer in solution with the substrate. In the caseof a macromer solution, the reactive units of the macromer may either bepresent at the end groups, or spaced throughout the backbone of themacromer in a random, block, or gradient fashion.

Preferably, the backbone coating is polymerised from constituentmonomers to provide an alternating or block copolymer. The alternating,or substantially alternating character, of the copolymer is believed toprovide an important spatial arrangement of the passive and activeconstituents which facilitates good surface coating of the substrate.Those skilled in the art will understand the degree of regularitynecessary in order for a copolymer to be considered of alternatingcharacter. It is preferred that the alternating copolymer has analternating character defined by greater than 70% of consecutivecomonomer residue units being alternate between residues of the firstcomonomer and the second comonomer, more preferably greater than 90%.The block nature of the copolymer may also vary in an alternatingfashion.

Preferably, the backbone coating is a copolymer of maleic anhydride andstyrene.

The Spacer

The spacer group provides a synthetic “handle” by which functionalgroups may be attached to the active constituent of the backbonecoating.

As used herein, the term “spacer,” “spacer molecule” and “spacer unit”are used interchangeably. As used herein, the term “functional tether”is used to refer to the combined moiety of a spacer molecule modifiedwith the desired functional group for the synthon.

In one preferred embodiment, the spacer molecule may be represented bythe generic structure shown in Scheme 2:

Generally, both X and Y comprise chemical moieties or substituent groupsthat may be chemically modified independently, sequentially or underorthogonal conditions. For example, X may chemically react with theactive constituent A to attach the spacer to the backbone. Subsequently,Y may be chemically modified with a desired functional group F.

Typical species may include for example, spacer molecules wherein X isthe residue of an amino, hydroxyl, thiol, carboxylic acid, anhydrides,isocyanate, sulfonyl chloride, sulfonic anhydride, chloroformate,ketone, or aldehyde; Y is the same as defined for X; and Q is a linearor branched divalent organic group; and X and Y are not reactive witheach other or Q. Preferably Q is selected from optionally substituted C₁to C₂₀ alkylene, optionally substituted C₂ to C₂₀ alkenylene, optionallysubstituted C₃ to C₂₀ cycloalkylene, optionally substituted C₂ to C₂₀alkynylene and optionally substituted C₆ to C₂₀ arylene, wherein one ormore carbon atoms may be substituted with a heteroatom selected from O,S or N.

By “optionally substituted” is meant that a group may or may not befurther substituted with one or more groups selected from, but notlimited to, alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl,haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy,haloalkoxy, haloalkenyloxy, acetyleno, carboximidyl, haloaryloxy,isocyano, cyano, formyl, carboxyl, nitro, nitroalkyl, nitroalkenyl,nitroalkynyl, nitroaryl, alkylamino, dialkylamino, alkenylamino,alkynylamino, arylamino, diarylamino, benzylamino, imino, alkylimine,alkenylimine, alkynylimino, arylimino, benzylimino, dibenzylamino, acyl,alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy,alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, heterocycloxy,heterocyclamino, haloheterocyclyl, alkylsulphonyl, arylsulphonyl,alkylsolphinyl, arylsulphinyl, carboalkoxy, alkylthio, benzylthio,acylthio, sulphonamido, sulfanyl, sulfo and phosphorus-containinggroups, alkoxysilyl, silyl, alkylsilyl, alkylalkoxysilyl, phenoxysilyl,alkylphenoxysilyl, alkoxyphenoxysilyl, arylphenoxysilyl, allophanyl,guanidino, hydantoyl, ureido, and ureylene. A carbon atom is consideredto be substituted if it has a double bond to a heteroatom, such asoxygen, sulfur or nitrogen to form a carbonyl, thiocarbonyl or iminegroup, respectively.

In the above definitions the terms “aryl” and “heteroaryl” refer to anysubstituent which includes or consists of one or more aromatic orheteroaromatic ring respectively, and which is attached via a ring atom.The rings may be mono or polycyclic ring systems, although mono orbicyclic 5 or 6 membered rings are preferred. Examples of suitable ringsinclude but are not limited to benzene, biphenyl, terphenyl,quaterphenyl, naphthalene, tetrahydronaphthalene, 1-benzylnaphthalene,anthracene, dihydroanthracene, benzanthracene, dibenzanthracene,phenanthracene, perylene, pyridine, 4-phenylpyridine, 3-phenylpyridine,thiophene, benzothiophene, naphthothiophene, thianthrene, furan,benzofuran, pyrene, isobenzofuran, chromene, xanthene, phenoxathiin,pyrrole, imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, indole,indolizine, isoindole, purine, quinoline, isoquinoline, phthalazine,quinoxaline, quinazoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, phenazine, isothiazole,isooxazole, phenoxazine and the like, each of which may be optionallysubstituted.

In the above definitions, the term “alkyl”, used either alone or incompound words such as “alkenyloxyalkyl”, “alkylthio”, “alkylamino” and“dialkylamino” denotes straight chain, branched or cyclic alkyl,preferably C₁₋₁₀ alkyl or cycloalkyl. Examples of straight chain andbranched alkyl include methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, sec-amyl,1,2-dimethylpropyl, 1,1-dimethyl-propyl, hexyl, 4-methylpentyl,1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl,2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl,1,3-dimethylbutyl, 1,2,2,-trimethylpropyl, 1,1,2-trimethylpropyl,heptyl, 5-methoxyhexyl, 1-methylhexyl, 2,2-dimethylpentyl,3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl,1,3-dimethylpentyl, 1,4-dimethyl-pentyl, 1,2,3,-trimethylbutyl,1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl,1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6-or 7-methyl-octyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, 1-,2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl,1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6-or 7-ethylnonyl, 1-, 2-, 3-, 4- or 5-propyloctyl, 1-, 2- or3-butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-,9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-ethyldecyl, 1-,2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl,1-2-pentylheptyl and the like. Examples of cyclic alkyl include mono- orpolycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and thelike.

In the above definitions the term “alkoxy” denotes straight chain orbranched alkoxy, preferably C ₁₋₁₀ alkoxy. Examples of alkoxy includemethoxy, ethoxy, n-propoxy, isopropoxy and the different butoxy isomers.

The term “alkenyl” denotes groups formed from straight chain, branchedor cyclic alkenes including ethylenically mono-, di- or poly-unsaturatedalkyl or cycloalkyl groups as previously defined, preferably C₂₋₁₀alkenyl. Examples of alkenyl include vinyl, allyl, 1-methylvinyl,butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl,1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl,3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl,1-decenyl, 3-decenyl, 1,3-butadienyl, 1-4,pentadienyl,1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl,1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl,1,3,5-cycloheptatrienyl and 1,3,5,7-cyclooctatetraenyl.

The term “alkynyl” denotes groups formed from straight chain, branchedor cyclic alkyne including those structurally similar to the alkyl andcycloalkyl groups as previously defined, preferably C₂₋₁₀ alkynyl.Examples of alkynyl include ethynyl, 2-propynyl and 2- or 3-butynyl.

The term “acyl” either alone or in compound words such as “acyloxy”,“acylthio”, “acylamino” or “diacylamino” denotes carbamoyl, aliphaticacyl group and acyl group containing an aromatic ring, which is referredto as aromatic acyl or a heterocyclic ring which is referred to asheterocyclic acyl, preferably C₁₋₁₀ acyl. Examples of acyl includecarbamoyl; straight chain or branched alkanoyl such as formyl, acetyl,propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl,2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl,decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl,pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyland icosanoyl; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl,t-butoxycarbonyl, t-pentyloxycarbonyl and heptyloxycarbonyl;cycloalkylcarbonyl such as cyclopropylcarbonyl, cyclobutylcarbonyl,cyclopentylcarbonyl and cyclohexylcarbonyl; alkylsulfonyl such asmethylsulfonyl and ethylsulfonyl; alkoxysulfonyl such as methoxysulfonyland ethoxysulfonyl; aroyl such as benzoyl, toluoyl and naphthoyl;aralkanoyl such as phenylalkanoyl (e.g. phenylacetyl, phenylpropanoyl,phenylbutanoyl, phenylisobutylyl, phenylpentanoyl and phenylhexanoyl)and naphthylalkanoyl (e.g. naphthylacetyl, naphthylpropanoyl andnaphthylbutanoyl; aralkenoyl such as phenylalkenoyl (e.g.phenylpropenoyl, phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl andphenylhexenoyl and naphthylalkenoyl (e.g. naphthylpropenoyl,naphthylbutenoyl and naphthylpentenoyl); aralkoxycarbonyl such asphenylalkoxycarbonyl (e.g. benzyloxycarbonyl); aryloxycarbonyl such asphenoxycarbonyl and napthyloxycarbonyl; aryloxyalkanoyl such asphenoxyacetyl and phenoxypropionyl; arylcarbamoyl such asphenylcarbamoyl; arylthiocarbamoyl such as phenylthiocarbamoyl;arylglyoxyloyl such as phenylglyoxyloyl and naphthylglyoxyloyl;arylsulfonyl such as phenylsulfonyl and napthylsulfonyl;heterocycliccarbonyl; heterocyclicalkanoyl such as thienylacetyl,thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl,thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl;heterocyclicalkenoyl such as heterocyclicpropenoyl,heterocyclicbutenoyl, heterocyclicpentenoyl and heterocyclichexenoyl;and heterocyclicglyoxyloyl such as thiazolylglyoxyloyl andthienylglyoxyloyl.

In alternative embodiments, the spacer molecule may have a branchedstructure whereby multiple functional groups may be attached at the endsof the branches.

Generally, there are two ways in which the spacer S may be incorporatedinto the synthon:

-   -   (1) A spacer molecule with a desired functional group already        attached to at least one end is chemically coupled to the        backbone.    -   (2) A spacer molecule is attached to the active constituent.        Then in a separate synthetic step, the spacer molecule is        further modified to attach a desired functional group.

In some embodiments, a spacer molecule may be attached, then modifiedwith more than one functional group.

In one embodiment the spacer molecule is a linear chain molecule and afunctional tether is formed by modifying the end of the chain distalfrom the site of attachment to the active constituent of the synthon.

By modifying the chemical or structural properties of the spacermolecule it is possible to generate synthons with a range of macroscopiccoating properties. For example, glycol oligomer chains provide arelatively rigid linear structure, whereas simple hydrocarbons adoptmore folded conformations. These differences in spacer geometry also mayvary with chain length or the presence of charged groups in the spacermolecule. These differences in geometry provided by the spacer moleculeproperties directly affects the orientation of the functional group withrespect to the backbone and thereby affects the overall macroscopicproperties of the surface coating. Modification of these properties maygreatly affect the complementary or antagonistic interactions betweenthe surface and a biomolecule, cell or other chemical entity immobilizedthereon.

Scheme 3 below illustrates the formation of a backbone coating on asubstrate and subsequent attachment of a spacer.

In Scheme 3, the backbone coating is applied by polymerization of theactive constituent, maleic anhydride, and the passive constituent,styrene. The spacer unit features an amine at one end that forms acovalent linkage to the active constituent resulting in a maleimide.

Preferably the spacer unit is a residue of a diamine, more preferably analkyl diamine. It is particularly preferred that the spacer unit S is aresidue of 1.5-diaminopentane or N-(3-aminopropyl)-1,3-propanediamine.

The Functional Group

The functional group may serve different roles in various embodiments.For example, the functional group may act as a site for further chemicalmodification of the surface. In the instance, where the functional groupis capped with a polymerization initiator, the possibility exists to addanother level of synthon diversity.

In Scheme 4 below, a spacer with an amine moiety provides the site forchemical modification with four different functional groups therebyresulting in four different, but related synthon surface coatings.

Preferably, the functional group F is a group capable of binding orchemically reacting with a biological molecule or component. Thefunctional group F also preferably comprises a primary or secondaryamine group.

Screening for Surface Optimization

In scheme 4, the functional group on each of the four coatings may serveas the primary site for a complementary binding interaction. Byscreening the four coatings in a desired solid phase binding assay, onemay determine which surface is optimal. Subsequently, based on the bestof the four synthons shown in Scheme 4, new libraries of relatedsynthons may be generated to further optimize the surface for thedesired application in an iterative fashion. For example, the nextiteration may vary only the spacer length. Hence, synthons may begenerated with functional groups exhibiting a range of moleculardiversity in order to find the optimal surface for binding acomplementary molecular species such as a receptor or other largebiomolecule. For example, a library of synthons may be generatedcomprising a range of functional groups in order to find the optimalsurface coating for binding the β-adrenergic receptor in a surfaceplasmon resonance experiment.

High-Throughput Advantage

Morever, Scheme 4 illustrates the high-throughput advantage afforded bysome embodiments of the synthon-based approached. As mentioned in theBackground of the Invention, generation of surface diversity on solidphases has been limited by the difficulty of developing chemical methodsfor grafting new coatings onto solid substrates. Prior methods havefocused on utilizing solution reactions to generate a diverse library ofcandidate compounds for coating a substrate. These methods haveencountered a bottleneck in getting the solution-phase compounds coatedonto a solid-phase substrate. This bottleneck results from the generallack of development of the science of grafting materials onto solids toform coatings.

As shown in Scheme 4, the present invention provides a high-throughputsolution to generating surface diversity by avoiding this bottleneck.Instead, in preferred embodiments, libraries of diverse surfaces may begenerated from a single backbone coating applied by a well-characterizedgrafting procedure. Subsequently, diversity may be introduced to thesolid phase surface in a combinatorial manner by varying the spacer andfunctional groups structures through well-known synthetic routes.

High-throughput generation of molecular diversity for detectingcomplementary binding interactions, as well as, for further chemicalmodification may be achieved by modifying the functional group on arelatively simple synthon backbone-spacer configuration. As shown inSchemes 5 and 6 below, when H₂N—S¹—X is a symmetrical diamine such asH₂N—(CH₂)₆—NH₂, a large number of functional groups with a range offunctional and molecular diversity may be added.

Incorporation of Grafting and Polymerization Methods

In a preferred embodiment, the synthon-based approach to generation ofdiverse surface coatings may be carried out using well-known orreadily-constructed free radical polymerization technology. Thisembodiment is particularly well-suited to generating synthon surfacecoatings on polymeric substrates such as polyolefins. In preferredembodiments, the polymeric substrate such as polypropylene or, may bealready be coated with sytreneic, (meth)acrylic, (meth)acrylamides, orother related graft coatings. The manner by which this initial coatingis a generated is well known in the art, gamma grafting, where by theinitiation requirements for the graft polymerisation to occur is from acobalt-60 source, or the like.

The Substrate

The combinatorial advantages of the present synthon-based surfacediscovery system are independent of the nature of the base substratematerial or how the synthon is applied to the surface. Hence surfacediversity may be explored across a wide range of substrates. Thesubstrate used in accordance with present invention is generally a solidand provides an integral surface or plurality of surfaces upon which thedifferent surface coating(s) may be applied. Preferably, the substrateis selected from glass, silicon, metals, and organic polymers, othersynthetic or natural materials, and combinations thereof.

The substrate may for example be provided in the form of a microscopeslide, microtitre plate, porous membrane, pipette tip, tube or aplurality of beads.

Preferably, the substrate is an organic polymer. Suitable organicpolymers include, but are not limited to, polytetrafluoroethylene,polystyrene, polypropylene, polyethylene, polyvinylidenefluoride andpolymethylmethacrylate.

Further, the substrate may be porous, non-porous, and/or any geometricshape, e.g. bead, or flat. A variety of porous polymeric substrates withco-continuous architecture useful with the present invention aredescribed in co-pending U.S. patent application Ser. No. 10/052,907filed Jan. 17, 2002, which is hereby incorporated by reference herein.

In a preferred embodiment of the invention the substrate is an organicpolymer in the form of a plurality of beads. Preferably, the beads arelabelled such that a particular coating can be related to a particularbead or subgroup of beads. Suitable polymeric beads for use as asubstrate in accordance with the present invention include, but is notlimited to, Luminex™ beads.

Multiplexed Applications

The present compositions and methods allow surface diversity to beexplored in a high-throughput fashion by, for example, buildingdifferent synthons in an array format on a single substrate. A varietyof multiplex formats such as arrays or beads may be used. For example, asingle synthon backbone coating may be applied across the full substratesurface. Then different spacer units or functional group variants may begenerated in different localized regions on the substrate.

As used herein, a “region” of a substrate includes a point, area orother location on the surface of the substrate. Each different surfacecoated on the substrate occupies discrete regions on the substrate.

In one preferred embodiment, photolithographic or micromirror methodsmay be used to spatially direct light-induced chemical modifications ofspacer units or functional groups resulting in attachment at specificlocalized regions on the surface of the substrate. Light-directedmethods of controlling reactivity and immobilizing chemical compounds onsolid substrates are well-known in the art and described in U.S. Pat.Nos. 4,562,157, 5,143,854, 5,556,961, 5,968,740, and 6,153,744, and PCTpublication WO 99/42813, each of which is hereby incorporated byreference herein.

Alternatively, plural localized synthon generation on a single substratemay be achieve by precise deposition of chemical reagents. Methods forachieving high spatial resolution in depositing small volumes of aliquid reagent on a solid substrate are disclosed in U.S. Pat. Nos.5,474,796 and 5,807,522, both of which are hereby incorporated byreference herein.

The term “array” may or may not require the identification of eachdifferent surface coating in terms of co-ordinates for its location. Anarray may be in a pattern or be random and may comprise two or morecoatings, or the same coating in different regions on the samesubstrate. The underlying substrate may be uniform in its ability toaccept a surface coating. Or the substrate may have regions withdifferent abilities to bind specific surface coatings resulting in aspatial pattern depending on the coating.

Screening of Diverse Surface Environments

Surface coatings prepared using the synthon-based approach of thepresent invention may find use in a wide range of solid phaseapplications. The generation of a combinatorial selection of surfacecoatings provides a spectrum of molecular and macroscopic surfaceproperties. The method provides a diversity of surface environments asshown in Scheme 7 below:

Each of these surfaces may potentially create an optimum environment orhave optimal properties for a particular solid phase application.However, the greater the number of diverse surfaces in a libraryrequires more screening for each particular application.

Generally, the surface coatings of the present invention may be screenedfor optimal performance in a solid phase application of interest bymethods well known in the art. For example, such screening may involvedetecting specific binding of cells to the surface and consequently mayutilize flow cytometry as, for example, described by Needels et al.(1993).

Other screening methods useful with the present invention include any ofthe great number of isotopic and non-isotopic labeling and detectionmethods well-known in the chemical and biochemical assay art. Forexample, a library of surface coatings of the present invention may bescreened for the ability to bind a specific peptide in an activeconfiguration on the surface. An active configuration refers to anorientation of the molecule on the surface coating whereby the moleculemay be specifically detected with a selected probe molecule, e.g. afluorescently coupled antibody that specifically binds the molecule.

Alternatively, spectroscopic methods well-known in the art may be usedto determine directly whether a molecule is bound to a surface coatingin an desired configuration. Spectroscopic methods include e.g., UV-VIS,NMR, EPR, IR, Raman, mass spectrometry and other methods adapted tosurface analysis well-known in the art.

Examples of biological compounds that may be screened for binding in theproper configuration on surface coating generated by the synthon-basedapproach of the present invention include, e.g. agonists and antagonistsfor cell membrane receptors, toxins, venoms, viral epitopes, hormones,sugars, cofactors, peptides, enzyme substrates, drugs inclusive ofopiates and steroids, proteins including antibodies, monoclonalantibodies, antisera reactive with specific antigenic determinants,nucleic acids, lectins, polysaccharides, cellular membranes andorganelles.

In addition, the present invention may be employed to generate optimalsurface coatings for immobilized nucleic acids. These coatings may beused in any of a large number of well-known hybridization assays wherenucleic acids are immobilized on a surface of a substrate, e.g.genotyping, polymorphism detection, gene expression analysis,fingerprinting, and other methods of DNA- or RNA-based sample analysisor diagnosis.

Various aspects of the present invention may be conducted in anautomated or semi-automated manner, generally with the assistance ofwell-known data processing methods. Computer programs and other dataprocessing methods well known in the art may be used to storeinformation including e.g. surface coating library chemical andmacroscopic properties. Data processing methods well known in the artmay be used to read input data covering the desired characteristics.

Alternatively, or in addition, data processing methods well known in theart may be used to control the processes involved in the presentinvention, including e.g applying or polymerizing the backbone coatingon the substrate; control of chemical reactions involved in furthergenerating the synthon; and/or the reactions and interactions occurringin, within or between a population or array of surface coatings on asubstrate.

The invention will now be described with reference to non-limitingexamples. However it is to be understood that the particularity of thefollowing description of the invention is not to supersede thegenerality of the invention previously described.

EXAMPLES

1. Generation of a Maleic Anhydride (MAn)-Styrene Backbone Coating on aPolymeric Solid Substrate.

Scheme 8, above, illustrates the reaction carried out in generating thebackbone coating. Plastic hollow cylinders, measuring 6mm in length, 3mm in diameter were pre-radiated in air at room temperature (1.8 KGy/hfor 7 hours). A 40% (v/v) solution of styrene and maleic anhydride,present in mole equivalent proportions, in toluene was prepared and theadded to the irradiated plastic cylinders. The mixture was then purgewith nitrogen gas for 5 minutes via a septum and heated, with agitationat 60° C. for 6 hours. The plastic cylinders were then isolated from thepolymerised solution, washed thoroughly to remove non-grafted polymerand dried to constant weight.

2. Attachment and Subsequent Deprotection of Tert-Butyl Carbamate (BOC)Protected Diamines Spacer Units to MAn-Sty Backbone Coating.

Step 1: Ring Opening with Amines (see Scheme 9, below)

A 1:1 DMF/Dioxane solution comprising an excess equivalents of theprotected diamine was charged with plastic cylinders prepared above inexample 1. A 6x excess DIEA was added to the solution and the solutionleft to react at 60 C for 2 hours, after which the plastic cylinderswhere isolated from the reaction mixture and washed thoroughly.Spectroscopic evidence (ATR and Raman) established the disappearance ofthe anhydride.

Step 2: Ring closure to the Imide (see Scheme 10, below)

The ring closure of the amic acid was effected by heating the materialfrom step 1 of example 2 prepared above, at 60 C in DMF in the presenceof acetic anhydride and sodium acetate for 4 hours. The plasticcylinders were then washed extensively to afford the ring closed,grafted imide.

Step 3: Liberating the Amine (see Scheme 11, below)

The removal of the amine protection group was performed under standardacid deprotection conditions by placing a sample of the plasticcylinders prepared above in example 2, step 2 were placed in a 20%Trifluoroacetic acid in dichloromethane for 2 hours. The deprotected,acidified samples were than washed extensively with dichloromethaneprior to neutralization.

Step 4: Neutralization of grafted amine (see Scheme 12, below)

The acidified samples prepared above in example 2, step 3 were treatedwith 5% triethyl amine in a 1:1 dimethyl formamide/dichloromethane, for20 minutes, then washed extensively with dimethyl formamide anddichloromethane, prior to drying and determination of amine activity asdescribed in Example 3, below.

3. Determination of Amine Activity.

A sample of the grafted material prepared above in example 2, step 4,were treated with an excess of Fmoc-β-Ala-OH in dichloromethane, in thepresence of diisopropyl carbodiimide. The Fmoc from the coupledFmoc-β-Ala-OH to the pendant amine on the plastic cylinder was thencleaved by exposure of the plastic cylinders to a 20% solutionpiperidine in dimethyl formamide and the liberated Fmoc detectedspectrophotometrically, to afford a concentration of active amines onthe graft of 0.108 micromoles.

4. Synthon Coating: Disks Examples

I. Library of Malcimides

Step 1. Preparation of Maleic Anhydride/Styrene Graft Co-polymer on PFAdisks. Maleic anhydride/Styrene was covalently attached onto atetrafluoroethylene-perfluoroalkyl-vinylether copolymer (PFA) disk usingthe γ-irradition technique. Three thousand PFA disks (6 mm diameter×0.8mm thickness) were immersed in 150 mL 20% maleic anhydride in ethylacetate (w/v) and 150 mL 20% styrene in ethyl acetate (v/v) containing0.010 M HCl in dioxane in a 500 mL glass bottle. The solution wasdegassed by bubbling with N_(2(g)) for 10 min. The glass bottle wassealed with a Teflon screw cap and γ-irradiated with a ⁶⁰Co source. Thegrafted disks were thoroughly washed with DMF and CH₂Cl₂ to removeresidual monomer and non-grafted co-polymer and dried overnight undervacuum at 30° C. After drying, the disks were weighed to give an averagemass change of 0.92% per disk (1.94 μg/mm²)

Step 2. Reaction of Maleic Anhydride/Styrene Graft System with PrimaryAmines.

A 50 mL glass vial was charged with maleic anhydride/styrene grafted PFAdisks (100 disks) and 20 mL of primary amine (1 M, Table 3) in DMFbefore the vial was sealed and shaken overnight. After 16 h, thesolution was removed and the disks washed with DMF and CH₂Cl₂ beforedrying under vacuum to give the mixed (amide-carboxylic acid-phenyl)system. TABLE 3 List of Primary Amines for Disks No. Amine 12-(Aminomethyl)-18-crown-6 2 4-METHOXYPHENETHYLAMINE 3 Benzylamine 4N-Acetylethylenediamine 5 Undecyclamine 6 1-NAPHTHALENEMETHYLAMINE 71-(2-AMINOETHYL)PYRROLIDINE 8 2-(2-Aminoethoxy)ethanol 9Tetrahydrofurfuryl amine 10 2-(2-CHLOROPHENYL)ETHYLAMINE 11 Propylamine12 2-(aminomethyl)pyridine 13 3,4-DIMETHOXYPHENETHYLAMINE 143-PHENYL-1-PROPYLAMINE 15 4-CHLOROBENZYLAMINE 161-(2-AMINOETHYL)PIPERIDINE 17 4-PHENYLBUTYLAMINE 18 4-AMINO-1-BUTANOL 194-FLUOROBENZYLAMINE 20 6-AMINO-1-HEXANOL 21 DECYLAMINE 22 NONYLAMINE 23Octylamine 24 VERATRYLAMINE 25 CYCLOHEXANEMETHYLAMINE 265-AMINO-1-PENTANOL 27 ISOPentylamine 28 1-(3-AMINOPROPYL)IMIDAZOLE 292-Methoxyethylamine 30 Ethanol amine 31 3-Aminopropionitrile 323-Methoxypropylamine 33 3-FLUOROBENZYLAMINE 343,4,5-Trimethoxybenzylamine 35 4-Methoxybenzylamine 362-Amino-1-propene-1,1,3-tricarbonitrile 37p-Aminophenyl-beta-D-glucopyranoside 38 D-Glucosamine hydrochloride 39p-Aminophenyl-beta-D-galactopyranoside 40 Bis-homotris 413-(Diethylamino)propylamine 42 2-METHOXYBENZYLAMINE 43 Isobutylamine 44BUTYLAMINE 45 4- (TRIFLUOROMETHYL)BENZYLAMINE 463,5-DIMETHOXYBENZYLAMINE 47 3-FLUOROPHENETHYLAMINE 48 Pentylamine

Step 3: Cyclization of Mixed System to give Styrene/Maleimide GraftCo-polymer. Mixed amide-carboxylic acid-styrene PFA disks (50 disks)derived from primary amines were treated with toluene (50 mL), aceticanhydride (0.25 M), and sodium acetate (0.025 M) before heating to 80°C. overnight. After 16 h, the vial was drained of reagent and the diskswashed with toluene, DMF, and then CH₂Cl₂ before drying under vacuum toafford the library of styrene/maleimide surfaces, generated from oneinitial surface.

At each stage in the coating assembly, XPS and ATR spectra were acquiredand indicated that each transformation had been performed. Further, theassembled library of maleimides on disks was screened against antiRabbit IgG, and a spectrum of very low to very high protein bindingsevents were observed.

II. Library of Maleimides with Diamine Spacers and Capping Groups

Step 1. Preparation of Maleic Anhydride/Styrene Graft Co-polymer on PFAdisks. Maleic anhydride/Styrene was covalently attached onto atetrafluoroethylene-perfluoroalkyl-vinylether copolymer (PFA) disk usingthe y-irradition technique. Three thousand PFA disks (6 mm diameter×0.8mm thickness) were immersed in 150 mL 20% maleic anhydride in ethylacetate (w/v) and 150 mL 20% styrene in ethyl acetate (v/v) containing0.010 M HCl in dioxane in a 500 mL glass bottle. The solution wasdegassed by bubbling with N_(2(g)) for 10 min. The glass bottle wassealed with a Teflon screw cap and γ-irradiated with a ⁶⁰Co source. Thegrafted disks were thoroughly washed with DMF and CH₂Cl₂ to removeresidual monomer and non-grafted co-polymer and dried overnight undervacuum at 30° C. After drying, the disks were weighed to give an averagemass change of 0.92% per disk (1.94 μg/mm²).

Step 2: Reaction of Maleic Anhydride/Styrene Graft System with Diamineson Disk

1943 PFA discs grafted with maleic anhydride/styrene from Step I werethen split into 29 batches of 67 discs. Each batch was treated with adifferent diamine (0.5 M in DMF) from Table 4 to give, after washing, 29different mixed (amide-carboxylic acid-phenyl) intermediates containingfree amines. TABLE 4 List of Diamine Spacers for Maleimide Library No.Diamine 1 Ethylenediamine 2 1,4-Diaminobutane 3 1,12-Diaminododecane 41,5-Diaminopentane 5 1,3-Diaminopropane 6 Diethylenetriamine 7Dipropylenetriamine 8 Tetraethylenepentamine 9 Triethylenetetramine 101,3-Cyclohexanebis(methylamine) 11 1,9-Diaminononane 124,9-Dioxa-1,12-dodecanediamine 13 N,N′-Bis(3-aminopropyl)ethylenediamine 14 Bis(hexamethylene)triamine 15Tris(2-aminoethyl)amine 16 Pentaethylenehexamine 171,4-Bis(3-aminopropyl)piperazine 18 2,2′-Oxybis(ethylamine)dihydrochloride 19 3,3′-Diamino-N- methyldipropylamine 202,2′-Dimethyl-1,3-diaminopropane 21 N,N′-Bis(2-aminoethyl)-1,3-propanediamine 22 2,2′-(Ethylenedioxy)bis(ethylamine) 23 DAB((PA)4Generation 1.0 24 DAB((PA)4 Generation 2.0 25 p-Xylylenediamine 26O,O′-Bis(3- aminopropyl)polyethylenediamine 27 Polyethylenimine 281,7-Diaminoheptane 29 4,7,10-Trioxa-1,13-trideanediamine

Step 3: Reaction of Mixed (Amide-carboxylic acid-phenyl) AmineIntermediates with Carboxylic Acids.

Each batch of diamines from step 2 was split into 67 different separatediscs. Each disc was treated with a different carboxylic acid from Table5 in a TABLE 5 List of Carboxylic Acid Capping Groups for MaleimideLibrary No. Acid 1 BOC-3-(1-naphthyl)-L-alanine 2 N(alpha)-BOC-L-lysine(Fmoc) 3 D-Tyrosine 4 O-tert-Butyl-L-serine (Fmoc) 5 FMOC-L-glutamicacid 5-benzyl ester 6 D-Phenylalanine (BOC) 7 BOC-L-Tyrosine 8L-Tyrosine (BOC) 9 N-Benzyloxycarbonyl-L-tyrosine 10FMOC-L-Phenylalanine 11 N-(9- FLUORENYLMETHOXYCARBONYL)-L- PROLINE 12N-alpha-Carbobenzyloxy-L-tryptophan 13 N-CBZ-L-METHIONINE 14N-FMOC-(L-ALANINE-OH)-H2O 15 N-Carbobenzyloxy-L-proline 162-(DIPHENYLPHOSPHINO)BENZOIC ACID 17 1-Pyrenebutyric acid 18(1S)-(−)-CAMPHANIC chloride 19 2,3,4,5-Tetrafluorobenzoyl chloride 20Docosanoic acid 21 2,6-Difluorophenylacetic acid 22 Piperonyloylchloride 23 2,3,4-TRIHYDROXYBENZOIC ACID 24 Pentafluorobenzoyl chloride25 4- METHOXYCYCLOHEXANECARBOXYLIC ACID 26 3-Iodo-4-methylbenzoic acid27 4-Octyloxybenzoic acid 28 Cyanoacetic acid 29 2-METHYL-1-CYCLOHEXANECARBOXYLIC ACID 30 N-TRITYLGLYCINE 31 3-Phenoxybenzoic acid32 3-Indolebutyric acid 33 3,5-Diisopropylsalicylic acid 344-Methylvaleric acid 35 2-Norbornane acetic acid 362,3,4-Trimethoxybenzoic acid 37 2-HYDROXY-1-NAPHTHOIC ACID 38 4-TERT-BUTYLCYCLOHEXANECARBOXYLIC ACID 39 2-Thiopheneacetic acid 402-Biphenylcarboxylic acid 41 3,4-Diaminobenzoic acid 42DIETHYLPHOSPHONOACETIC ACID 43 Flufenamic acid 44 TRIDECANOIC ACID 45(1R,3R,4R,SR)-(−)-QUINIC ACID 46 2,2-Bis(hydroxymethyl)propionic acid 47p-Toloyl chloride 48 Propionic anhydride 49 3-Mercaptopropionic acid 50Gibberellic acid 51 Z-L-leucyl-L-alanine 52R(+)-N-(alpha-Methylbenzyl)phthalic acid monoamide 53(+)-mono-(1S)-Menthyl phthalate 54 R(−)-2-Oxothiazolidine-4-carboxylicacid 55 9H-Fluorene-9-carboxylic acid 56 Orotic acid anhydrous 57BOC-L-leucine 58 15-Hydroxypentadecanoic acid 59 ACEMETACIN 60N-T-BOC-S-TRITYL-L-CYSTEINE 61 URACIL-4-ACETIC ACID 62(+/−)-4-METHYLOCTANOIC ACID 63 N-ALPHA-T-BOC-NEPSILON-CBZ-L- LYSINE 64Indomethacin 65 N-BENZOYL-BETA-ALANINE 66 N-ACETYL-L-TRYPTOPHAN 67MEFENAMIC ACIDsolution of DMF, 1-hydroxy-7-azabenztriazole (0.25 M), anddiisopropylethylamine (0.5 M). The reaction was agitated overnightbefore washing with DMF and methylene chloride to remove excess reagent.

Step 4: Cyclization of Mixed System to give Styrene/Maleimide GraftCo-polymer. Mixed amide-carboxylic acid-styrene PFA disks from step 3(50 disks) were treated with acetic anhydride (0.25 M) and sodiumacetate (0.025 M) in toluene before heating to 80° C. overnight. After16 h, the vial was drained of reagent and the disks washed with toluene,DMF, and then CH₂Cl₂ before drying under vacuum to afford the library ofstyrene/maleimide surfaces, generated from one initial surface.

At each stage in the coating assembly, XPS and ATR spectra were acquiredand indicated that each transformation had been performed. Further, theassemble library of maleimides with diamine spacers and dapping groupson disks was screened against anti Rabbit IgG, and a spectrum of verylow to very high protein bindings were observed.

5. Synthon Coating: Microarray Examples

I. Library of Maleimides

Step 1. Preparation of Maleic Anhydride/Styrene Graft Co-polymer onmicroscope slide.

A procedure for applying a Synthon Coating in a microarray format can beaccomplished as follows: A microscope slide of dimensions 2.5×7.5×0.1cm, prepared from the injection molding oftetrafluoroethylene-perfluoroalkyl-vinylether copolymer (PFA), can bemasked to create an array of 16×250 urn circular spots. Treatment of themasked slide with heptane plasma (5 min, 20 W, 10⁻³ torr) followed byremoval of the mask yields a PFA slide consisting of 16×250 urn thinlycoated heptane spots. UV irradiation of the slide in the presence ofbenzophenone (0.05 M) in methanol followed by simultaneouspolymerization and grafting of maleic anhydride (1.75 M) and styrene(1.75 M) in ethyl acetate selectively derivatizes the heptane layer togive arrayed spots that are densely functionalised with anhydridegroups.

Step 2. Reaction of Maleic Anhydride/Styrene Graft Slide with PrimaryAmines.

Primary amine containing compounds (0.5 M) dissolved in DMF readilyattach to the surface upon robotic printing of nanolitre droplets toeach spot via ring opening of the anhydride. Each spot of 3 slides fromstep I were treated with a different primary amine (Table 6) to givethree microarrays of 16 different mixed (amide-carboxylic acid-phenyl)intermediates. The arrays were washed exhaustively with DMF, CH₂Cl₂, and1% acetic acid in DMF before drying under vacuum. TABLE 6 List ofPrimary Amines for Microarray No. Amine 1 2-(Aminomethyl)-18-crown-6 24-METHOXYPHENETHYLAMINE 3 Benzylamine 4 N-Acetylethylenediamine 5Undecyclamine 6 1-NAPHTHALENEMETHYLAMINE 7 1-(2-AMINOETHYL)PYRROLIDINE 82-(2-Aminoethoxy)ethanol 9 Tetrahydrofurfuryl amine 102-(2-CHLOROPHENYL)ETHYLAMINE 11 Propylamine 12 2-(aminomethyl)pyridine13 3,4-DIMETHOXYPHENETHYLAMINE 14 3-PHENYL-1-PROPYLAMINE 154-CHLOROBENZYLAMINE 16 1-(2-AMINOETHYL)PIPERIDINE 17 4-PHENYLBUTYLAMINE18 4-AMINO-1-BUTANOL 19 4-FLUOROBENZYLAMINE 20 6-AMINO-1-HEXANOL 21DECYLAMINE 22 NONYLAMINE 23 Octylamine 24 VERATRYLAMINE 25CYCLOHEXANEMETHYLAMINE 26 5-AMINO-1-PENTANOL 27 ISOPentylamine 281-(3-AMINOPROPYL)IMIDAZOLE 29 2-Methoxyethylamine 30 Ethanol amine 313-Aminopropionitrile 32 3-Methoxypropylamine 33 3-FLUOROBENZYLAMINE 343,4,5-Trimethoxybenzylamine 35 4-Methoxybenzylamine 362-Amino-1-propene-1,1,3-tricarbonitrile 37p-Aminophenyl-beta-D-glucopyranoside 38 D-Glucosamine hydrochloride 39p-Aminophenyl-beta-D-galactopyranoside 40 Bis-homotris 413-(Diethylamino)propylamine 42 2-METHOXYBENZYLAMINE 43 Isobutylamine 44BUTYLAMINE 45 4-(TRIFLUOROMETHYL)BENZYLAMINE 46 3,5-DIMETHOXYBENZYLAMINE47 3-FLUOROPHENETHYLAMINE 48 Pentylamine

Step 3: Cyclization of Mixed System to give Styrene/Maleimide GraftCo-polymer.

Subsequent dehydration of the entire array using acetic anhydride (0.25M) and sodium acetate (0.025 M) at 80° C. in toluene gives arrays of 16different surface bound maleimides/styrene co-polymers.

At each stage in the coating assembly, XPS and ATR spectra were acquiredand indicated that each transformation had been performed. Further, theassemble library of maleimides on a microarray was screened against antiRabbit IgG, and a spectrum of very low to very high protein bindingsevents were observed.

II. Library of Mixed (amide-carboxylic acid-phenyl) Systems fromSecondary Amines on Microarray.

Step 1. Preparation of Maleic Anhydride/Styrene Graft Co-polymer onmicroscope slide.

A procedure for applying a Synthon Coating in a microarray format can beaccomplished as follows: A microscope slide of dimensions 2.5×7.5×0.1cm, prepared from the injection molding oftetrafluoroethylene-perfluoroalkyl-vinylether copolymer (PFA), can bemasked to create an array of 16×250 um circular spots. Treatment of themasked slide with heptane plasma (5 min, 20 W, 10⁻³ torr) followed byremoval of the mask yields a PFA slide consisting of 16×250 urn thinlycoated heptane spots. UV irradiation of the slide in the presence ofbenzophenone (0.05 M) in methanol followed by simultaneouspolymerization and grafting of maleic anhydride (1.75 M) and styrene(1.75 M) in ethyl acetate selectively derivatizes the heptane layer togive arrayed spots that are densely functionalised with anhydridegroups.

Step 2. Reaction of Maleic Anhydride/Styrene Graft Slide with SecondaryAmines.

A PFA slide grafted with 16 maleic anhydride/styrene spots waselaborated with 16 different secondary amines (0.5 M, Table 7) dissolvedin DMF via robotic printing. Washing of the slide with dimethylformamidefollowed by 1% acetic acid in dimethylformamide gives 16×250 umdifferent mixed (amide-carboxylic acid-styrene) spots on the PFA slide.TABLE 7 List of Secondary Amines for Microarray No. Secondary Amine 1Dimethylamine 2 3,3-Iminodipropionitrile 3 Morpholine 4Bis(2-methoxyethyl)amine 5 Piperidine 6 Diethyl amine 7N-Benzylmethylamine 8 1-Methylpiperazine 9 4-Piperidinone monohydratehydrochloride 10 1-Acetylpiperazine 11 1,2,3,4-Tetrahydroisoquinoline 12Pyrrolidinone 13 N-Methylpropargyl amine 14N,N,N′-Trimethylethylenedianine 15 Thiomorpholine 16 Nipecotamide

At each stage in the coating assembly, XPS and ATR spectra were acquiredand indicated that each transformation had been performed. Further, theassemble library of mixed (amide-carboxylic acid-phenyl) systems fromsecondary amines on microarray was screened against anti Rabbit IgG, anda spectrum of very low to very high protein bindings events wereobserved.

III. Library of Mixed (amide-amide-phenyl) System on Microscope Slide

Step 1. Preparation of Maleic Anhydride/Styrene Graft Co-polymer onmicroscope slide.

A procedure for applying a Synthon Coating in a microarray format can beaccomplished as follows: A microscope slide of dimensions 2.5×7.5×0.1cm, prepared from the injection molding oftetrafluoroethylene-perfluoroalkyl-vinylether copolymer (PFA), can bemasked to create an array of 16×250 um circular spots. Treatment of themasked slide with heptane plasma (5 min, 20 W, 10⁻³ torr) followed byremoval of the mask yields a PFA slide consisting of 16×250 um thinlycoated heptane spots. UV irradiation of the slide in the presence ofbenzophenone (0.05 M) in methanol followed by simultaneouspolymerization and grafting of maleic anhydride (1.75 M) and styrene(1.75 M) in ethyl acetate selectively derivatizes the heptane layer togive arrayed spots that are densely functionalised with anhydridegroups.

Step 2: Reaction of Maleic Anhydride/Styrene Graft Slide with SecondaryAmines.

A PFA slide grafted with 16 maleic anhydride/styrene spots waselaborated with 16 different secondary amines (0.5 M, Table 7 above)dissolved in DMF via robotic printing. Washing of the slide withdimethylformamide followed by 1% acetic acid in dimethylformamide gives16×250 un different mixed (amide-carboxylic acid-styrene) spots on thePFA slide.

Step 3. Reaction of Mixed (amide-carboxylic acid-phenyl) System withDiamine.

Twenty-nine copies of the slide in step 2 were treated with DMAP (10 mol%), 1,3-diisopropyl carbodiimide (0.25 M), and N-hydroxysuccinimide(0.15M) in DMF. After washing with DMF, the slides were separated andeach treated with a different diamine from Table 6 above. After severalhours, the slides were washed with DMF and allowed to dry under vacuumto give microarrays of mixed (2°-Amide-1°-amide-phenyl)amine systems.Hence, all slides contain the same 16 secondary amines, one for eachspot, but each slide contains a different diamine, wherein all spots ona given slide have the same diamine.

Step 4: Reaction of Mixed (2°-Amide-1°amide-phenyl) Amine Intermediateswith Carboxylic Acids.

The thirty slides from step 3 above were each treated with a solution of3-iodo-4-methylbenzoic acid (0.25 M), 1-hydroxy-7-azabenztriazole (0.25M), and diisopropylethylamine (0.5 M) in DMF. The reaction mixtures wereagitated overnight before washing with DMF and methylene chloride toremove excess reagent.

At each stage in the coating assembly, XPS and ATR spectra were acquiredand indicated that each transformation had been performed. Further, theassemble library of mixed (amide-amide-phenyl) system on a microarraywas screened against anti Rabbit IgG, and a spectrum of very low to veryhigh protein bindings events were observed.

6. Synthon Coating: Carboxylated Polymer Bead Examples

Synthon Coating Polymer

Inhibitor free styrene (86.4 mmol), maleic anhydride (86.4 mmol), andinitiator AIBN (0.1 mmol) were mixed together in 1,4-Dioxane (48 ml) ina polymerisation ampoule and sealed with a rubber septum. The solutionwas degassed by nitrogen sparging then allowed to polymerise at 60° C.in a temperature controlled oil bath. After an appropriate time intervalthe polymerisation was stopped by precipitation into a 10-fold excess ofmethanol. The copolymer was collected by filtration and purified once byreprecipitation into methanol from DMF. The alternating copolymer wascharacterised my GPC: Mw=270 000.

0.5 grams of the afforded polymer was dispersed into 50 ml of Milliporewater and hdrolyzed at 80° C. with shaking over 5 days to afford theSynthon Coating Polymer, that is employed in the bead and plate examplesbelow.

A) Absorption of the Synthon Coating Polymer

Step 1: A 100 uL bead suspension of 5 micron, carboxylated was washedonce with 2 mls of Millipore water. The suspension was spun down and thebead plug resuspended into 1 ml of a 1 wt % solution of PEI (Aldrich,750K). The PEI was allowed to adsorb for 30 minutes with occasionalgentle shaking and subsequently washed vigorously 3 times with Milliporewater and spun down to a bead plug. The PEI coated beads were thenresuspended in 1 ml of 1% hydrolysed Synthon Coating Polymer 1(described above) and allowed to adsorb for 30 min with occasionalgentle shaking. The beads were then washed 3 times with Millipore waterwith each washing step including 20 min of gentle shaking and spun downto a bead plug.

Step 2: To effect the next coating stage, the spun down bead plugs withthe PEI and adsorbed Synthon Coating Polymer were resuspended into 1 mlof a 5 mg/ml EDC water solution and after 1 min, 25 uL of the 1,5 pentyldiamine was added. The samples were shaken briefly and the couplingreaction was allowed to proceed for 2 hrs with occasional gentleshaking. As the beads tended to clump during this process, they wereredispersed with a short stints in the ultrasonic bath. The diaminecoupled beads were then washed exhaustively with Millipore water 5 timesand spun down to a bead plug. These amine modified beads wereresuspended into 1 ml of water and 200 uL of the,3-iodo-4-methyl-benzoic acid, sulfo-NHS ester (˜10 mg/ml of DMF) wasadded. The reaction was left to proceed for 2 hrs and were thenexhaustively washed 5 times with Millipore water. It should be notedthat this modification can be effected by any number of diamines (orother multi-amine building block) and carboxylic acids, to allow thegeneration of libraries of modified encoded beads from the singleSynthon Coating Polymer modified bead.

At each stage in the coating assembly, XPS spectra were acquired andindicated that each transformation had been performed. This process wasperformed on a number of beads sets from Bangs Laboratories (L020621N,L020325G& Dyed: L011009A) and Luminex (L100-C124-01)

B) Covalent Attachment of the Synthon Coating Polymer

Step 1: A 100 uL bead suspension of 5 micron, carboxylated was washedonce with 2 mls of Millipore water. The suspension was spun down and thebead plug resuspended into 1 ml of a 1 wt % solution of PEI (Aldrich,750K). The PEI was allowed to adsorb for 30 minutes with occasionalgentle shaking and subsequently washed vigorously 3 times with Milliporewater and spun down to a bead plug. The covalent attachment of theSynthon Coating Polymer to the PEI coated beads was performed byresuspending the PEI beads in 1 ml of 1% Synthon Coating Polymer(preparation described above) that had been activated with EDC, and thereaction allowed to proceed for 30 min with occasional gentle shaking.The beads were then washed 3 times with Millipore water with eachwashing step including 20 min of gentle shaking and spun down to a beadplug.

Step 2: To effect the next coating stage, the spun down bead plugs withthe PEI and adsorbed Synthon Coating Polymer were resuspended into 1 mlof a 5 mg/ml EDC water solution and after 1 min, 25 uL of the 1,5 pentyldiamine was added. The samples were shaken briefly and the couplingreaction was allowed to proceed for 2 hrs with occasional gentleshaking. As the beads tended to clump during this process, they wereredispersed with a short stints in the ultrasonic bath. The diaminecoupled beads were then washed exhaustively with Millipore water 5 timesand spun down to a bead plug. These amine modified beads wereresuspended into 1 ml of water and 200 uL of the,3-iodo-4-methyl-benzoic acid, sulfo-NHS ester (˜10 mg/ml of DMF) wasadded. The reaction was left to proceed for 2 hrs and were thenexhaustively washed 5 times with Millipore water. It should be notedthat this modification can be effected by any number of diamines (orother multi-amine building block) and carboxylic acids, to allow thegeneration of libraries of modified encoded beads from the singleSynthon Coating Polymer modified bead.

At each stage in the coating assembly, XPS spectra were acquired andindicated that each transformation had been performed. This process wasperformed on a number of beads sets from Bangs Laboratories (L020621N,L020325G& Dyed: L011009A) and Luminex (L100-C124-01)

C) Multiplex Bead Based Assay

Encoded Carboxylated beads employed in the assay were acquired fromLuminex, and treated with Step 1 of the Absorption of the SynthonCoating Polymer described above. 5.0×10⁶ microspheres were transferredto a 15 mL microcentrifuge tube, spun down to a pellet and resuspendedin 5 mL of 0.1 M MES, pH 4.5 making sure to vortex and sonicate beadswell.

0.2 nmol of capture oligo probes (2 mL of 1:10 of stock in dH20) wasadded to the beads, followed by a fresh aliquot of 10 mg/mL EDC in dH20(2.5 mL). The reaction was allowed to proceed for 30 minutes at roomtemperature in the dark, prior to washing and charging the vessel withanother fresh solution of 2.5 mL of EDC. This solution was alsoincubated for 30 minutes at room temperature in the dark, then washedwith 1.0 mL of 0.02% Tween-20. The suspension was centrifuged for 1minute to produce pellet and the supernatant carefully removed. Thebeads were then washed with 1.0 mL of 0.1% SDS, centrifuged for 1 minuteto produce pellet and the supernatant carefully removed. The beads werethen finally suspended in 100 mL of TE, at pH 8.0 and stored at 2-8° C.in complete darkness.

The coupled beads were then resuspended 1.5× TMAC buffer and distributedto a sample or background well on the PCR plate. The amplifiedbiotinylated DNA was then added and TE, pH 8.0 added to make a total of1 7 mL. The solutions were gently pipet up and down to mix. The sampleswere covered with plate sealer and place in thermocycler under a programthat is set at 95° C. (denaturing step) for 5 minutes and then 52° C.(hybridization step) for 15 minutes. The plate was then spun (32250×g, 3minutes) and the supernatant carefully removed, and the plate placedback into the PCR at 52° C. 75 mL of reporter solution was then added toeach well, mixed gently and incubate at 52° C. for 5 minutes prior toanalysis via a Luminex machine, to afford an improved signal to noiseover the non-modified Encoded Carboxylated beads.

7. Coating of a Multi-well Plate

A) Non-Reactive Microtitre Plate

Step 1: 200 uL of a 1 wt % PEI (Aldrich, 750K) was added to the wells ofa 96 well microtitre plate (Maxisorp, Nunc) and allowed to stand at roomtemperature for 60 min. The wells were then washed 5 times withMillipore water. 200 uL of a 1 wt % Synthon Coating Polymer (preparationdescribed above) was added to the wells and the interaction allowed toproceed for 60 min. The wells were then washed 5 times with Milliporewater.

Step 2: 200 uL of a 5 vol % 1,5 pentyl diamine in 5 mg/ml EDC watersolution was added to the wells and coupling allowed to proceed for 2hrs, and then the wells were washed 5 times with Millipore water. 200 uLof a coupling solution comprising 5 mg/ml EDC and 5 mg/ml3-iodo-4-methyl-benzoic acid in DMSO was added to the wells and allowedto proceed for 2 hours after which the wells were washed twice withfresh DMSO then 5 times with Millipore water.

It should be noted that this modification can be effected by any numberof diamines (or other multi-amine building block) and carboxylic acids,to allow the generation of libraries of modified microtitre plate wellsfrom a single Synthon Coating Polymer modified bead.

At each stage in the coating assembly, XPS spectra were acquired andindicated that each transformation had been performed. The modifiedplates could then be employed in standard immunoassay protocols forELISA and other diagnostic procedures

B) Reactive Microtitre Plate

Step 1: 200 uL of a 1 wt % Synthon Coating Polymer (preparationdescribed above) was added to the wells NHS active plate, DNA-BIND(Coming) and ReactiBind plate (Piece) and the reaction allowed toproceed for 60 min. The wells were then washed 5 times with Milliporewater.

Step 2: 200 uL of a 5 vol % 1,5 pentyl diamine in 5 mg/ml EDC watersolution was added to the wells and coupling allowed to proceed for 2hrs, and then the wells were washed 5 times with Millipore water. 200 uLof a coupling solution comprising 5 mg/ml EDC and 5 mg/ml3-iodo-4-methyl-benzoic acid in DMSO was added to the wells and allowedto proceed for 2 hours after which the wells were washed twice withfresh DMSO then 5 times with Millipore water.

It should be noted that this modification can be effected by any numberof diamines (or other multi-amine building block) and carboxylic acids,to allow the generation of libraries of modified microtitre plate wellsfrom a single Synthon Coating Polymer modified bead.

At each stage in the coating assembly, XPS spectra were acquired andindicated that each transformation had been performed. The modifiedplates could then be employed in standard immunoassay protocols forELISA and other diagnostic procedures.

8. Coating of PVDF Membrane

Step 1: Activation of the Membrane with a Grafted Synthon Polymer

Four, 10×20 cm pieces of Immobilon-P^(SQ) PVDF membrane (Millipore) wereplaced into a 700 ml beaker. The beaker was filled with a 1.5M ethylacetate solution of 1:1 Styrene and Maleic anhydride, degassed bynitrogen purging and sealed. The solution was then irradiated in a gammacell for 100 min. The irradiated membranes were removed from thepolymerisation solution and washed with a large excess of ethyl acetate.Once washing was complete, the membranes were dried under high vacuumovernight and stored in a low humidity cupboard.

Step 2. Modification of the Membrane

A standard solution of the amine in THF (100 ml, 0.25 M, 0.025 mol) wasprepared for each amine used. Grafted PVDF membranes were cut to a sizeof 10×10 cm, and placed in a large Petrie dish. The 100 ml aminesolution was then carefully poured into the Petri dish, ensuring thatthe membrane was fully wet. The Petri dishes were then sealed with lidsand allowed to agitate (very slowly) overnight at room temperature. Thereaction solution was removed from the petri dish and the membraneswashed with THF, dried under vacuum overnight and stored in the lowhumidity cupboard.

It should be noted that this modification can be effected by any numberof amines (or other multi-amine building block) to allow the generationof libraries of modified PVDF membranes from a single grafted SynthonPolymer modified membrane.

At each stage in the coating assembly, XPS and ATR spectra were acquiredand indicated that each transformation had been performed. The modifiedplates could then be employed in standard electroblotting protocols forwestern blotting applications to increase the amount of captured proteinavailable for immunoassay.

9. Synthon Coating: Determining the Optimium Coating for a DesiredSpecific Application

Step 1: Preparation of Library on Desired Format:

A library of different but related surfaces are assembled in the desiredformat (microarray, bead, plated, etc) for the application, employingthe methods described above.

Step 2: Screening of the Assembled Library

The assembled libraries are screened against the desired target for thedesired application such as a biological screen for kinases, Rabbit IgG,cytokines or a synthetic screen for reaction optimizations, or the like.The outcome from this screen would be to identify the optimum surfacefor the said desired application, in a rapid and cost effective manner.

If the desired level of signal is not attained from the first screen ofthe libraries, a second, more focused library is then assembled with theknowledge from the first and the screen repeated until the desired levelof signal is obtained. More than one surface from each screen may afforda signal of the desired level.

Step 3: Generation of a Synthon coating for a Desired SpecificApplication.

Having determined the optimum surface for the desired application, theidentified surface can then be assembled by any means required, thataffords the surface in a timely and cost effective mariner. Further, theoutcomes of a number of screening events can be assembled onto onesurface, such as a microarray, resulting in a multiplex platform having,or consisting of multiple elements or parts to do more than oneexperiment.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications which fall within itsspirit and scope. The invention also includes all the steps, features,compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations of any two or more of said steps or features.

1. A method of generating a library of different surface coatings on asubstrate comprising: a) selecting a surface coating synthon of formulaB—S—F, wherein B is a copolymer of at least one passive constituent Pand at least one active constituent A, S is a spacer unit and F is achemical or biological functional group, wherein S is attached to anactive constituent A of copolymer B, and wherein the synthon has atleast one point of diversity selected from P, A, S and F; b) applyingbackbone coating(s) of the selected copolymer B onto a substrate; c)attaching the selected combination(s) of spacer unit S and functionalgroup F to an active constituent A of copolymer B according to saidselected synthon; wherein steps b) and c) are performed such thatsurface coatings according to the synthon are generated on localisedregions of the substrate, thereby providing said library of differentsurface coatings on the substrate.
 2. The method according to claim 1,wherein the substrate is selected from an organic polymer, glass,silicon, metal and combinations thereof.
 3. The method according toclaim 1, wherein the substrate is in the form of a microscope slide,microtitre plate, porous membrane, pipette tip, tube or a plurality ofbeads.
 4. The method according to claim 2, wherein the substrate is anorganic polymer.
 5. The method according to claim 4, wherein the organicpolymer is selected from polytetrafluoroethylene, polystyrene,polypropylene, polyethylene, polyvinylidenefluoride andpolymethylmethacrylate.
 6. The method according to claim 4, wherein theorganic polymer is in the form of a plurality of beads.
 7. The methodaccording to claim 6, wherein the beads are labelled such that aparticular coating can be related to a particular bead or subgroup ofbeads.
 8. The method according to claim 7, wherein the beads areLuminex™ beads.
 9. The method according to claims 1, wherein the librarycomprises at least 10 different surface coatings.
 10. The methodaccording to claims 1, wherein the library comprises at least 100different surface coatings.
 11. The method according to claims 1,wherein the library comprises at least 1,000 different surface coatings.12. The method according to claims 1, wherein the library comprises atleast 10,000 different surface coatings.
 13. The method according toclaims 1, wherein the active constituent A of copolymer B is apolymerised residue of a compound selected from those listed in Table 1of this specification.
 14. The method according to claim 13, wherein theactive constituent A of copolymer B is a polymerised residue of maleicanhydride.
 15. The method according to claims 1, wherein the passiveconstituent P of copolymer B is a polymerised residue of a compoundselected from those listed in Table 2 of this specification.
 16. Themethod according to claim 15, wherein the passive constituent P ofcopolymer B is a polymerised residue of styrene.
 17. The methodaccording to claims 1, wherein copolymer B is an alternating copolymer.18. The method according to claims 1, wherein copolymer B is a blockcopolymer of the active constituent A and the passive constituent P. 19.The method according to claims 1, wherein copolymer B is a copolymer ofmaleic anhydride and styrene.
 20. The method according to claims 1,wherein copolymer B further comprises a control agent C.
 21. The methodaccording to claim 20, wherein the control agent is selected from a RAFTcontrol agent, an ARTP control agent and a nitroxide control agent. 22.The method according to claims 1, wherein the spacer unit S has thestructure:X-Q-Y wherein X is the residue of an amino, hydroxyl, thiol, carboxylicacid, anhydride, isocyanate, sulfonyl chloride, sulfonic anhydride,chloroformate, ketone or aldehyde moiety; Y is the same as defined forX; and Q is a divalent organic group, and wherein X and Y are notreactive with each other or Q.
 23. The method according to claim 22,wherein Q is selected from optionally substituted C₁ to C₂₀ alkylene,optionally substituted C₂ to C₂₀ alkenylene, optionally substituted C₂to C₂₀ alkynylene and optionally substituted C₆ to C₂₀ arylene, whereinone or more carbon atoms may be substituted with a heteroatom selectedfrom O, S or N.
 24. The method according to claim 22, wherein the spacerunit S is a residue of a diamine.
 25. The method according to claim 24,wherein the spacer unit S is a residue of an alkyl diamine.
 26. Themethod according to claim 25, wherein the spacer unit S is a residue of1,5-diaminopentane or N-(3-aminopropyl)-1,3-propanediamine.
 27. Themethod according to claims 1, wherein the chemical or biological group Fis a group capable of binding or chemically reacting with a biologicalmolecule or component.
 28. The method according to claim 27, wherein thechemical or biological group F comprises a primary or secondary aminegroup.
 29. The method according to claims 1, wherein the synthon has,within the active constituent A, the passive constituent P, the spacerunit S and the functional group F, a sole point of diversity in theselection of the spacer unit S.
 30. The method according to claims 1,wherein the synthon has, within the active constituent A, the passiveconstituent P, the spacer unit S and the functional group F, a solepoint of diversity in the selection of the functional group F.
 31. Themethod according to claims 1, wherein the synthon has, within the activeconstituent A, the passive constituent P, the spacer unit S and thefunctional group F, two points of diversity in the selection of thespacer unit S and the functional group F.
 32. The method according toclaims 1, wherein the backbone coating(s) of copolymer B are appliedonto the substrate by grafting, or other methods of coating selectedfrom dip coating, plasma polymerisation, vapor deposition, stampprinting, gamma irradiation, electron beam exposure, and thermal andphotochemical radiation.
 33. The method according to claims 1, whereinthe selected combination(s) of spacer unit S and functional group F areattached by: 1) attaching the spacer unit S to copolymer B and thenattaching the functional group F to the attached spacer group S; or 2)attaching the spacer unit S to copolymer B, wherein the spacer unit Salready has the functional group F attached to it.
 34. The methodaccording to claims 1, wherein the backbone coating(s) of selectedcopolymer B is applied onto localised regions of the substrate.
 35. Themethod according to claim 34, wherein the backbone coating(s) ofselected copolymer B is applied to a plurality of beads.
 36. The methodaccording to claims 1, wherein the backbone coating(s) of selectedcopolymer B is applied to the surface of the substrate, and the selectedcombination(s) of spacer unit S and functional group F are attached tothe copolymer B in localised regions.
 37. The method according to claims1, wherein the surface coatings according to the synthon which aregenerated on localised regions of the substrate are spatially resolved.38. A method of optimizing a substrate surface for a solid-phaseapplication involving immobilization of a molecule comprising: a)generating a library of different surface coatings on a substrate by amethod comprising: 1) selecting a surface coating synthon of formulaB—S—F, wherein B is a copolymer of at least one passive constituent Pand at least one active constituent A, S is a spacer unit and F is achemical or biological functional group, wherein S is attached to anactive constituent A of copolymer B, and wherein the synthon has atleast one point of diversity selected from P, A, S and F; 2) applyingbackbone coating(s) of the selected copolymer B onto a substrate; 3)attaching the selected combination(s) of spacer unit S and functionalgroup F to an active constituent A of copolymer B according to saidselected synthon; wherein steps 2) and 3) are performed such thatsurface coatings according to the synthon are generated on localisedregions of the substrate, thereby providing said library of differentsurface coatings on the substrate; b) exposing at least two of thesurface coatings in the library to the molecule to be immobilized; andc) determining which of the at least two surfaces results in betterperformance of the immobilized molecule in the solid-phase application.39. The method of claim 38 wherein the solid-phase application involvesimmobilization of a biological molecule or a biological molecule analogselected from proteins, peptides, peptide nucleic acids, nucleic acids,non-natural nucleic acids, oligonucleotides and carbohydrates.
 40. Themethod according to claim 38, wherein the solid-phase applicationinvolves detecting binding of a ligand to an immobilised biologicalmolecule.
 41. A solid phase application involving immobilisation of abiological molecule, wherein the biological molecule is immobilised on asubstrate surface optimized by the method of claim
 38. 42. A method oftailoring a surface coating to recognise, bind to or associate with aparticular biological molecule comprising: a) generating a library ofdifferent surface coatings on a substrate by a method comprising: 1)selecting a surface coating synthon of formula B—S—F, wherein B is acopolymer of at least one passive constituent P and at least one activeconstituent A, S is a spacer unit and F is a chemical or biologicalfunctional group, wherein S is attached to an active constituent A ofcopolymer B, and wherein the synthon has at least one point of diversityselected from P, A, S and F; 2) applying backbone coating(s) of theselected copolymer B onto a substrate; 3) attaching the selectedcombination(s) of spacer unit S and functional group F to an activeconstituent A of copolymer B according to said selected synthon; whereinsteps 2) and 3) are performed such that surface coatings according tothe synthon are generated on localised regions of the substrate, therebyproviding said library of different surface coatings on the substrate;b) exposing at least two of the surface coatings in the library to theparticular biological molecule; and c) determining which of the at leasttwo surfaces best recognises, binds to or associates with the particularbiological molecule.